SARS-CoV-2 variant of concern type and biological sex affect efficacy of molnupiravir in dwarf hamster model of severe COVID-19

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   ScreenIT

   Feb 10, 2022

   SciScore for 10.1101/2022.02.04.479171: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Experiments with SARS-CoV-2 involving ferrets and dwarf hamsters were approved by the Georgia State Institutional Animal Care and Use Committee under protocol A20031 and A21019, respectively.
   Sex as a biological variable	Lonza Bioscience, cat# CC-2540S, lot# 0000646466, passage 3, donor “M4”) from a 38-year-old male were expanded in PneumaCult-Ex Plus (Stemcell Technologies cat# 05040) and differentiated in PneumaCult-ALI (Stemcell Technologies cat# 05001) for 8 weeks in following the manufacturer’s instructions.
   Randomization	After a minimal resting period of 2 weeks after arrival, animals were randomly assigned to groups for individual studies, transferred into an ABSL-3 facility …

   SciScore for 10.1101/2022.02.04.479171: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Experiments with SARS-CoV-2 involving ferrets and dwarf hamsters were approved by the Georgia State Institutional Animal Care and Use Committee under protocol A20031 and A21019, respectively.
   Sex as a biological variable	Lonza Bioscience, cat# CC-2540S, lot# 0000646466, passage 3, donor “M4”) from a 38-year-old male were expanded in PneumaCult-Ex Plus (Stemcell Technologies cat# 05040) and differentiated in PneumaCult-ALI (Stemcell Technologies cat# 05001) for 8 weeks in following the manufacturer’s instructions.
   Randomization	After a minimal resting period of 2 weeks after arrival, animals were randomly assigned to groups for individual studies, transferred into an ABSL-3 facility immediately prior to study start, and housed singly in ventilated negative-pressure cages during the studies.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cells were authenticated and checked for mycoplasma prior to use.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies: SARS-CoV-2 N and goblet cells were co-stained using rabbit anti-SARS-CoV-2	anti-SARS-CoV-2 suggested: None
   Nucleocapsid monoclonal antibody (HL453) (Invitrogen, cat# MA5-36272) (1:100 dilution) and mouse anti-MUC5AC (ThermoFisher, cat# MA5-12175) (1:200 dilution) as primary antibodies, respectively.	HL453 suggested: (Thermo Fisher Scientific Cat# MA5-36272, RRID:AB_2890568) anti-MUC5AC suggested: (Thermo Fisher Scientific Cat# MA5-12175, RRID:AB_10983421)
   Donkey anti-goat (Alexa Fluor® 568 (ThermoFisher Scientific, cat# A-11057)) and rabbit anti-mouse IgG (H+L) cross-adsorbed secondary antibody (Alexa Fluor® 488 (ThermoFisher Scientific, cat# A-11059)) were used at a 1:500 dilution as secondary antibodies.	anti-goat suggested: (Molecular Probes Cat# A-11057, RRID:AB_142581)
   For staining of SARS-CoV-2 S, mouse anti SARS-CoV-1 and 2 Spike protein clone [1A9] monoclonal (Abcam, cat# ab273433) (1:200 dilution) and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor® 488; 1:500 dilution (Invitrogen, cat# A-11029)), were used as primary and secondary antibodies, respectively.	anti SARS-CoV-1 and 2 Spike protein suggested: (Abcam Cat# ab273433, RRID:AB_2891068) anti-mouse IgG suggested: (Molecular Probes Cat# A-11029, RRID:AB_2534088)
   For staining of ciliated cells, rabbit anti-beta IV tubulin recombinant antibody conjugated with Alexa Fluor® 647 [EPR16775] (Abcam, cat# ab204034) was used at a 1:100 dilution.	rabbit anti-beta IV tubulin recombinant antibody suggested: None anti-beta IV tubulin suggested: (Abcam Cat# ab179509, RRID:AB_2716759)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: African green monkey kidney cells VeroE6 (ATCC CRL-1586™),	VeroE6 suggested: None
   VOC delta (lineage B.1.617.2, clinical isolate #2333067) and VOC omicron (lineage B.1.1.529, WA-UW-21120120771) were obtained from the Northwestern Reference laboratory and amplified on Calu-3 cells.	Calu-3 suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
   For hamsters infected with VOC gamma, delta, and omicron, plaque assays were performed with VeroE6-TMPRSS2 cells.	VeroE6-TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Software and Algorithms
   Sentences	Resources
   Log viral titers were normalized using the average top plateau of viral titers to define 100% and were analyzed with a non-linear regression with variable slope to determine EC50 (Prism; GraphPad)	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Image captures were performed with a Zeiss Axio Observer Z.1 and an LSM 800 confocal microscope with AiryScan, controlled with the Zeiss Zen 3.1 Blue software package (Windows 10).	Zen suggested: None
   Statistics and reproducibility: The Microsoft Excel (versions 16.52) and Numbers (version 10.1) software packages were used for most data collection.	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137) Numbers suggested: (BioNumbers, RRID:SCR_002782)
   The GraphPad Prism (version 9.1.0) software package was used for data analysis.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Figures were assembled using Adobe Illustrator (version CS6).	Adobe Illustrator suggested: (Adobe Illustrator, RRID:SCR_010279)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2022.02.04.479171 on bioRxiv

   Feb 7, 2022